We present a conservation genetics tool kit, which offers two ready-to-use workflows for the routine application of genetic methods in conservation management. The workflows were optimized for work load and costs and are accompanied by an easy-to-read and richly illustrated manual with guidelines regarding sampling design, sampling of genetic material, necessary permits, laboratory methods, statistical analyses and documentation of results in a practice-oriented way. The manual also provides a detailed interpretation help for the implementation of the results in conservation management. One workflow deals with the identification of pond-breeding amphibians based on metabarcoding and environmental DNA (eDNA) from water samples. This workflow also discriminates the morphologically similar water frogs (Pelophylaxsp.) and other closely related species (e.g.Triturus cristatusandT. carnifex). The second workflow studies connectivity among populations using microsatellite markers. Its statistical analyses encompass the detection of genetic groups and historical, recent and current dispersal and gene flow. Using the two workflows does not involve academic research institutes; they can be applied by environmental consultancies, laboratories from the private sector, governmental agencies or non-governmental organisations. These and additional conservation genetic workflows will hopefully foster the routine use of genetic methods in conservation management.

Die Aufgabe, Gelder moglichst wirksam und am richtigen Ort einzusetzen, stellt im Naturschutz angesichts der beschrankten Mittel und des gleichzeitig grossen Handlungsbedarfs eine besondere Herausforderung dar. Problematisch ist beispielsweise, dass bei der Biodiversitatsforderung oftmals eine Wirkungskontrolle der Massnahmen schwierig oder mit grossem Aufwand verbunden ist. Die in den letzten Jahren zunehmend angewandten genetischen Methoden zum Nachweis von Arten sowie zur Erfassung von Vernetzung von Populationen und Lebensraumen haben gezeigt, dass die Anwendung der Genetik die Naturschutzpraxis bei verschiedenen Fragestellungen wesentlich unterstutzen kann. Noch werden aber genetische Methoden in der Naturschutzpraxis selten angewandt. Der neu entwickelte Werkzeugkasten Naturschutzgenetik bietet der Naturschutzpraxis Ablaufe beziehungsweise Workflows an, welche es erlauben genetische Methoden im Naturschutz routinemassig einzusetzen. Hauptbestandteil des Werkzeugkastens Naturschutzgenetik sind bezuglich Aufwand und Ertrag erprobte, einfache und transparente Workflows mit illustrierten Anleitungen fur das Probenahmedesign, das Sammeln von Proben im Feld, die Analysen im Labor, standardisierte statistische Auswertungen sowie die einfache benutzerorientierte Darstellung der Resultate und deren Interpretation zuhanden der Naturschutzpraxis (Bund, Kantone, Umweltburos, NGOs). Das gezielte Ausrichten auf fur den Naturschutz wichtige und relevante Fragestellungen tragt dazu bei, das Potential der genetischen Methoden fur den Alltag im Naturschutz nutzbar zu machen und die Naturschutzgenetik in der Praxis zu etablieren. Es wurden zwei Ablaufe oder Workflows fur den routinemassigen und standardisierten Einsatz von genetischen Methoden im Naturschutz eingefuhrt (fur aktuelle Informationen siehe www.naturschutzgenetik.ch). Erstens wurde ein genetischer Nachweis der in der Schweiz in Weihern und Teichen vorkommenden Amphibienarten aufgrund von eDNA (Umwelt-DNA) aus Wasserproben etabliert. Damit konnen auch Amphibienarten nachgewiesen werden, die mit herkommlichen Methoden nur schwer nachweisbar sind (z. B. Kammmolch, Triturus cristatus, Teichmolch, Lissotriton vulgaris) oder im Feld morphologisch nicht unterscheidbar sind (z. B. invasive Wasserfrosche, Pelophylax sp.). Zweitens wurde ein Workflow fur die Untersuchung von Vernetzung beziehungsweise Isolation von Populationen und Lebensraumen mittels genetischer Methoden (Mikrosatelliten) entwickelt. Zurzeit sind genetische Methoden die einzigen, welche allgemein und fur alle Organismengruppen mit vertretbarem Aufwand anwendbar sind, um Vernetzung auf Landschaftsebene festzustellen. Die beiden entwickelten Workflows sind so konzipiert, dass sie auf viele Ziellebensraume und Zielarten des Naturschutzes ubertragbar sind. Der Werkzeugkasten Naturschutzgenetik ist somit einfach erweiterbar. Bereits wird der Workflow eDNA aufgrund des Interesses von AnwenderInnen um weitere Organismengruppen (z. B. Libellen) erweitert. Mit dem Werkzeugkasten Naturschutzgenetik soll es gelingen, die Nutzung der Naturschutzgenetik mit ihren vielfaltigen Anwendungsmoglichkeiten in der Naturschutzpraxis in der Schweiz zu verankern.

Understanding the influence of landscape elements on movement and connectivity of potentially isolated populations is essential for successful conservation management, especially in agricultural landscapes. Here, we assessed the movement and genetic structure of the endangered Natterjack toad (Epidalea calamita) in an intensively managed agricultural landscape of the Central Plateau in Switzerland. Movement was analysed on the basis of molecular markers and radio-tracking, supplemented by long-term monitoring data describing population trends. A total of 19 migration events were detected by genetic first-generation assignment tests (11), by unintentional repeated genetic sampling of individuals (6) and by radio-tracking between predefined populations (2). None of the breeding sites was genetically isolated, although there was a trend that, in addition to geographic distances>2.5km, built-up areas enhanced genetic differentiation. Analysing the toad populations as a spatial network supported the inference that the two largest populations were sources, suggesting population size as a major driver of the movement pattern. We conclude that the Natterjack toad is well established in the intensely managed agricultural landscape, given that (i) large populations are maintained which may act as sources to spatially distributed occurrences, and (ii) the intensively managed landscape is sufficiently interspersed with suitable breeding habitats.

Microsatellite DNA families (MDF) are stretches of DNA that share similar or identical sequences beside nuclear simple‐sequence repeat (nSSR) motifs, potentially causing problems during nSSR marker development. Primers positioned within MDFs can bind several times within the genome and might result in multiple banding patterns. It is therefore common practice to exclude MDF loci in the course of marker development. Here, we propose an approach to deal with multiple primer‐binding sites by purposefully positioning primers within the detected repetitive element. We developed a new protocol to determine the family type and the primer position in relation to MDFs using the software packages repark and repeatmasker together with an in‐house R script. We re‐evaluated newly developed nSSR markers for the lepidopteran Marbled White (Melanargia galathea) and explored the implications of our results with regard to published data sets of the butterfly Euphydryas aurinia, the grasshopper Stethophyma grossum, the conifer Pinus cembra and the crucifer Arabis alpina. For M. galathea, we show that it is not only possible to develop reliable nSSR markers for MDF loci, but even to benefit from their presence in some cases: We used one unlabelled primer, successfully binding within an MDF, for two different loci in a multiplex PCR, combining this family primer with uniquely binding and fluorescently labelled primers outside of MDFs, respectively. As MDFs are abundant in many taxa, we propose to consider these during nSSR marker development in taxa concerned. Our new approach might help in reducing the number of tested primers during nSSR marker development.

Black poplar (Populus nigra L.) is a keystone species of riparian ecosystems in large parts of Europe. While adult black poplars were thoroughly surveyed across Europe, there is much less information about the species' natural regeneration. As the cultivar Populus x canadensis often grows in the surroundings of black poplar stands, introgression is being discussed as threat for black poplar rejuvenation. In the present study, we identified the proportion of black poplar seedlings and juvenile trees at three sites in Switzerland as compared to hybrid poplar offspring growing at the same sites. Taxon identification was based on a combination of a chloroplast and two nuclear markers. Using SSR markers, we estimated the extent of clonality of adult and juvenile trees, estimated genetic diversity of all age classes and determined the proportion of gene flow from outside the local stand. We found different proportions of black poplar regeneration at our three study sites, ranging from no hybrid offspring in Valais, where most adult black poplars relative to adult P. x canadensis occurred, to 90% hybrid seedlings in Thurgau, where the black poplar stand consisted of only four individuals, surrounded by a large number of P. x canadensis. The extent of clonality was generally low, as we identified less than 10% recurrent genotypes among juveniles and adults. We found high numbers of alleles and high heterozygosity in all population. Substantial gene flow from outside the local stands could be detected in two study sites, indicating that black poplar at least partly overcomes the current degree of fragmentation in Switzerland. (C) 2016 Elsevier GmbH. All rights reserved.

Changes in the forest management practices have strongly influenced the distribution of species inhabiting old-growth forests. The epiphytic woodland lichen Lobaria pulmonaria is frequently used as a model species to study the factors affecting the population biology of lichens. We sampled 252 L. pulmonaria individuals from 12 populations representing three woodland types differing in their ecological continuity and management intensity in Estonia. We used eight mycobiont-specific microsatellite loci to quantify genetic diversity among the populations. We calculated the Sørensen distance to estimate genetic dissimilarity among individuals within populations. We revealed that L. pulmonaria populations have significantly higher genetic diversity in old-growth forests than in managed forests and wooded meadows. We detected a significant woodland-type-specific pattern of genetic dissimilarity among neighbouring L. pulmonaria individuals, which suggests that in wooded meadows and managed forests dominating is vegetative reproduction. The vegetative dispersal distance between the host trees of L. pulmonaria was found to be only 15–30 m. Genetic dissimilarity among individuals was also dependent on tree species and trunk diameter. Lobaria pulmonaria populations in managed forests included less juveniles compared to old-growth forests and wooded meadows, indicating that forest management influences life stage structure within populations. We conclude that as intensive stand management reduces the genetic diversity of threatened species in woodland habitats, particular attention should be paid to the preservation of remnant populations in old-growth habitats. Within managed habitats, conservation management should target on maintenance of the stand’s structural diversity and availability of potential host trees.

The dwarf bulrush (Typha minima Funck ex Hoppe) is an endangered pioneer plant species of riparian flood plains. In Switzerland, only 3 natural populations remain, but reintroductions are planned. To identify suitable source populations for reintroductions, we developed 17 polymorphic microsatellite markers with perfect repeats using the 454 pyrosequencing technique and tested them on 20 individuals with low-cost M13 labeling. We detected 2 to 7 alleles per locus and found expected and observed heterozygosities of 0.05-0.76 and 0.07-1, respectively. The whole process was finished in less than 6 weeks and cost approximately USD 5000. Due to low costs and reduced expenditure of time, the use of next-generation sequencing techniques for microsatellite development represent a powerful tool for population genetic studies in nonmodel species, as we show in this first application of the approach to a plant species of conservation importance.

Habitat fragmentation has been generally regarded detrimental to the persistence of many species, especially those with limited dispersal abilities. Yet, when exactly habitat elements become functionally disconnected very much depends on the dispersal ability of a species in combination with the landscape's composition in which it occurs. Surprisingly, for many small and ground-walking generalists knowledge at what spatial scale and to what extent landscape structure affects dispersal is very scarce. Because it is flightless, the bush cricket Pholidoptera griseoaptera may be regarded susceptible to fragmentation. We applied habitat occupancy surveys, population genetic analyses and movement modelling to investigate the performance of P. griseoaptera in an agricultural mosaic landscape with suitable habitat patches of varying size and isolation. Despite its presumed dispersal limitation we could show that P. griseoaptera occupied the majority of suitable habitats, including small and isolated patches, showed a very low and non-significant genetic differentiation (F ST = 0.0072) and, in the model, managed to colonize around 73% of all suitable habitat patches within one generation under weak and strong landscape-effect scenarios. We conclude that P. griseoaptera possesses the behavioural attributes (frequent inter-patch dispersal) necessary to persist in this landscape characterized by a patchy distribution of habitat elements. Yet, sound recommendations to landscape planning and conservation require more research to determine whether this represents a general behaviour of the species or a behavioural adaptation to this particular landscape.

BACKGROUND AND AIMS:A previous study detected no allozyme diversity in Iberian populations of the buckler-fern Dryopteris aemula. The use of a more sensitive marker, such as microsatellites, was thus needed to reveal the genetic diversity, breeding system and spatial genetic structure of this species in natural populations.METHODS:Eight microsatellite loci for D. aemula were developed and their cross-amplification with other ferns was tested. Five polymorphic loci were used to characterize the amount and distribution of genetic diversity of D. aemula in three populations from the Iberian Peninsula and one population from the Azores.KEY RESULTS:Most microsatellite markers developed were transferable to taxa close to D. aemula. Overall genetic variation was low (H(T) = 0.447), but was higher in the Azorean population than in the Iberian populations of this species. Among-population genetic differentiation was high (F(ST) = 0.520). All loci strongly departed from Hardy-Weinberg equilibrium. In the population where genetic structure was studied, no spatial autocorrelation was found in any distance class.CONCLUSIONS:The higher genetic diversity observed in the Azorean population studied suggested a possible refugium in this region from which mainland Europe has been recolonized after the Pleistocene glaciations. High among-population genetic differentiation indicated restricted gene flow (i.e. lack of spore exchange) across the highly fragmented area occupied by D. aemula. The deviations from Hardy-Weinberg equilibrium reflected strong inbreeding in D. aemula, a trait rarely observed in homosporous ferns. The absence of spatial genetic structure indicated effective spore dispersal over short distances. Additionally, the cross-amplification of some D. aemula microsatellites makes them suitable for use in other Dryopteris taxa.

Populus nigra is considered a rare and threatened tree species in Switzerland because of dramatic habitat loss owing to river regulations during the last two centuries and because of potential gene introgression from non-indigenous P. deltoides through planted P. x canadensis hybrids. The significance of introgression as an endangerment to P. nigra, however, is controversial. The aims of the present study were (1) to assess how abundant P. nigra trees are in Switzerland and (2) to assess potential gene introgression. We present data from a molecular survey of 1372 putative P. nigra trees from Switzerland, using both chloroplast DNA and nuclear DNA markers. The results show that P. nigra is more abundant in Switzerland than hitherto thought. Furthermore, we detected a low frequency of gene introgression.

Small populations of self-incompatible plants are assumed to be threatened by a limitation of compatible mating partners due to low genetic diversity at the self-incompatibility (S) locus. In contrast, we show by using a PCR-RFLP approach for S-genotype identification that 15 small populations (N = 8-88) of the rare wild pear (Pyrus pyraster) displayed no mate limitation. S-allele diversity within populations was high (N = 9-21) as was mate availability (92.9-100%). Although population size and S-allele diversity were strongly related, no relationship was found between population size and mate availability, gene diversity (He), or fixation index (F(IS)), based on five neutral microsatellite loci. As we determined the principal mate availability within populations based on the S-genotypes observed, the realized mate availability under natural conditions may differ from our estimates, for example, due to spatially limited pollen dispersal. We therefore urge studies on self-incompatible plants to proceed from the simple assessment of principal mate availability to the determination of realized mate availability in natural populations.

Epiphytes are strongly affected by the population dynamics of their host trees. Owing to the spatio-temporal dynamics of host tree populations, substantial dispersal rates - corresponding to high levels of gene flow - are needed for populations to persist in a landscape. However, several epiphytic lichens have been suggested to be dispersal-limited, which leads to the expectation of low gene flow at the landscape scale. Here, we study landscape-level genetic structure and gene flow of a putatively dispersal-limited epiphytic lichen, Lobaria pulmonaria. The genetic structure of L. pulmonaria was quantified at three hierarchical levels, based on 923 thalli collected from 41 plots situated within a pasture-woodland landscape and genotyped at six fungal microsatellite loci. We found significant isolation by distance, and significant genetic differentiation both among sampling plots and among trees. Landscape configuration, i.e. the effect of a large open area separating two forested regions, did not leave a traceable pattern in genetic structure, as assessed with partial Mantel tests and analysis of molecular variance. Gene pools were spatially intermingled in the pasture-woodland landscape, as determined by Bayesian analysis of population structure. Evidence for local gene flow was found in a disturbed area that was mainly colonized from nearby sources. Our analyses indicated high rates of gene flow of L. pulmonaria among forest patches, which may reflect the historical connectedness of the landscape through gene movement. These results support the conclusion that dispersal in L. pulmonaria is rather effective, but not spatially unrestricted.

Dispersal is a process critical for the dynamics and persistence of metapopulations, but it is difficult to quantify. It has been suggested that the old-forest lichen Lobaria pulmonaria is limited by insufficient dispersal ability. We analyzed 240 DNA extracts derived from snow samples by a L. pulmonaria-specific real-time PCR (polymerase chain reaction) assay of the ITS (internal transcribed spacer) region allowing for the discrimination among propagules originating from a single, isolated source tree or propagules originating from other locations. Samples that were detected as positives by real-time PCR were additionally genotyped for five L. pulmonaria microsatellite loci. Both molecular approaches demonstrated substantial dispersal from other than local sources. In a landscape approach, we additionally analyzed 240 snow samples with real-time PCR of ITS and detected propagules not only in forests where L. pulmonaria was present, but also in large unforested pasture areas and in forest patches where L. pulmonaria was not found. Monitoring of soredia of L. pulmonaria transplanted to maple bark after two vegetation periods showed high variance in growth among forest stands, but no significant differences among different transplantation treatments. Hence, it is probably not dispersal limitation that hinders colonization in the old-forest lichen L. pulmonaria, but ecological constraints at the stand level that can result in establishment limitation. Our study exemplifies that care has to be taken to adequately separate the effects of dispersal limitation from a limitation of establishment.

1. To persist in a highly fragmented environment, such as the intensively used agricultural landscapes of Europe, species may need to successfully disperse between sub-populations. This paper describes a capture - mark - resight experiment to investigate movement and dispersal patterns of the flightless bush cricket species Pholidoptera griseoaptera.2. Movement parameters showed little difference between juveniles and imagos as well as between males and females. Both developmental stages and sexes moved considerable distances, were cap\able of crossing habitat boundaries, and showed successful inter-patch dispersal through the agricultural matrix.3. The results suggest that P. griseoaptera exhibits movement behaviours that compensate for its inability to fly. It was concluded that the good dispersal ability exhibited by animals of both sexes and developmental stage is an important factor allowing P. griseoaptera to persist in fragmented agricultural landscapes.